Chain-melting Transition Temperature Research Articles

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Effect of Anesthetics on Action Potential Propagation

Local anesthesia has been attributed to the specific interaction of local anesthetics with (sodium) channel proteins, while the action of general anesthetics still remains unclear. However, already at the beginning of 20th century Meyer and Overton independently found that the critical anesthetic dose of anesthetics, regardless of their types, is linearly proportional to their solubility in olive oil. Based on this finding, in 2005 Heimburg and Jackson proposed that the action potential is a density pulse (soliton) propagating in biological membranes, which explains anesthesia by freezing point depression law that originates from van’t Hoff's law. In this work, we conduct experiments on nerves from lobsters and earthworms to study the effect of anesthetics on compound action potential and action potential from a single neuron. The experimental data are then compared with the simulation results by solving the soliton equations in 1D cylindrical membrane with anesthetics inside the system. Anesthetics move the chain melting transition temperature of membranes far away from the physiological temperature, which requires a higher free energy to induce the phase transition, resulting in a higher stimulation voltage to reach the maximum amplitude of the action potential.

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60°C. PGPC forms 3.5nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, XPGPC≤0.3 and coexisting vesicles and micelles at higher XPGPC. Data suggest that liquid phase DPPC at 50°C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for XPGPC≤0.3. At 60°C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all XPGPC for T≥22°C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for XPGPC>0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for XPGPC≤0.3. For XPGPC≤0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.

Synthesis, physicochemical characterization and membrane interactions of a homologous series of N-acylserotonins: Bioactive, endogenous conjugates of serotonin with fatty acids

N-Acylserotonins (NASTs), present in the mammalian gastro-intestinal tract and central nervous tissues, exhibit significant biological and pharmacological activities. In the present study, a homologous series of NASTs have been synthesized and characterized. Differential scanning calorimetric studies show that in the dry and hydrated states the transition temperatures, enthalpies, and entropies of NASTs exhibit odd–even alternation. Both odd and even chain length NASTs independently display linear dependence of the transition enthalpies and entropies on the chain length under dry as well as hydrated conditions, suggesting that the molecular packing and intermolecular interactions in each series (odd or even) are likely to be similar for NASTs with different acyl chain lengths in the dry state as well as in the hydrated state. Powder X-ray diffraction studies indicated that the incremental increase in the d-spacing per CH2 group is 1.023Å, suggesting that the lipid acyl chains are most likely packed in an interdigitated fashion. Results of computational studies are consistent with this and suggest that the acyl chains of the NASTs are tilted with respect to the bilayer normal. Incorporation of N-myristoylserotonin (NMST) into dimyristoylphosphatidylcholine (DMPC) membranes did not significantly affect the phase transition properties at low mole fractions (1–5mol%), although distinct decrease in the chain-melting transition temperature and increase in the pretransition temperature were observed at higher contents (7.5–30mol%), suggesting that NMST increases the stability of the tilted gel phase (Lβ′) but destabilizes the ripple phase (Pβ′). These observations provide a thermodynamic basis for understanding the functional role of NASTs in their parent tissues.

Pore Formation in a Binary Giant Vesicle Induced by Cone-Shaped Lipids

We have investigated shape deformations of binary giant unilamellar vesicles (GUVs) composed of cone- and cylinder-shaped lipids. By coupling the spontaneous curvature of lipids with the phase separation, we demonstrated pore opening and closing in GUVs. When the temperature was set below the chain melting transition temperature of the cylinder-shaped lipid, the GUVs burst and then formed a single large pore, where the cone shape lipids form a cap at the edge of the bilayer to stabilize the pore. The pore closed when we increased the temperature above the transition temperature. The pore showed three types of shapes depending on the cone-shaped lipid concentration: simple circular, rolled-rim, and wrinkled-rim pores. These pore shape changes indicate that the distribution of the cone- and cylinder-shaped lipids is asymmetric between the inner and outer leaflets in the bilayer. We have proposed a theoretical model for a two-component membrane with an edge of bilayer where lipids can transfer between two leaflets. Using this model, we have reproduced numerically the observed shape deformations at the rim of pore.

Structural and thermodynamic determinants of chain-melting transition temperatures for phospholipid and glycolipids membranes

For optimum function, biological membranes need a fluid environment, which is afforded by the liquid-disordered phase of lipids with low chain-melting temperatures or the liquid-ordered phase that is formed by combining high chain-melting lipids with cholesterol. The dependence of chain-melting transition temperature on lipid chain structure is therefore of central importance. The currently available database, including sphingolipids and glycolipids, is summarised here by parameterising systematic dependences on molecular structure in terms of suitable thermodynamic models. Chain-length dependence, chain asymmetry of lipids forming partially interdigitated and mixed interdigitated gel phases, chain unsaturation, positional dependence of methyl branching, headgroup-attached and α-branched chains, and length of zwitterionic headgroups are all covered. This type of information is essential for biophysical approaches to functional lipidomics.

Pressure Perturbation Calorimetric Studies on Phospholipid-Sterol Mixtures

Sterols regulate biological processes and sustain the lateral structure of cellular membranes. The sterol cholesterol, its precursor lanosterol, the plant sterols stigmasterol and ergosterol as well as 7-dehydrocholesterol were added up to 36 mol-% to vesicles of the phospholipid 1,2-dipalmitoyl-snglycero- 3-phosphatidylcholine (DPPC). The aim of this study was to investigate the influence of the sterol side chain and ring structure on the volumetric properties of the lipid bilayer system by using pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the thermal expansion coefficient and volumetric properties of biomolecules. The experiments were carried out in the temperature range from 10 to 85 °C, i. e., at temperatures below and above the chain-melting transition temperatures of the lipid mixtures. Additionally, corresponding differential scanning calorimetric (DSC) measurements were carried out.Whereas the conformational properties of the different sterols have a significant effect on the order parameter of the lipid acyl-chains, the thermodynamic parameters of these sterols are less influenced by the differential structural changes of the sterols. For lanosterol and stigmasterol, marked differences are found, however.

Erratum to “Shifts in chain-melting transition temperature of liposomal membranes by polymer-grafted lipids” [Biochim. Biophys. Acta 1614 (2003) 165–170

Erratum to “Shifts in chain-melting transition temperature of liposomal membranes by polymer-grafted lipids” [Biochim. Biophys. Acta 1614 (2003) 165–170

Shifts in chain-melting transition temperature of liposomal membranes by polymer-grafted lipids

The chain-melting transition temperature of dipalmitoyl phosphatidylcholine (DPPC) bilayer membranes containing poly(ethylene glycol)-grafted dipalmitoyl phosphatidylethanolamine (PEG-DPPE) was determined by optical turbidity measurements. The dependence on content, X p, of PEG-DPPE lipid was studied for different polar headgroup sizes, n p, of the polymer lipid, throughout the lamellar phase of the mixtures with DPPC. Mean-field theory for the polymer brush regime predicts that the downward shift in transition temperature should vary with polymer size and content as n p X p 5/3 (∼ n p X p 11/6 for scaling theory). Any shift induced by the charge on PEG-lipids is independent of polymer size. These predictions are reasonably borne out for the longer polymer lipids (PEG molecular masses 750, 2000 and 5000 Da). Transition temperature shifts in the lamellar phase, before the onset of micellisation, are in the region of −1 to −2 °C (±0.1–0.2 °C) in reasonable accord with theoretical estimates of the lateral pressure exerted by the polymer brush. Shifts of this size are significant to the design of liposomes for controlled release of contents by mild hyperthermia.

La 3+ stabilizes the hexagonal II (H II) phase in phosphatidylethanolamine membranes

The mechanism of the effects of the lanthanum ion (La 3+) and the gadolinium ion (Gd 3+), which are lanthanides, on the function of membrane proteins and the stability of the membrane structure is not well understood. We investigated the effects of La 3+ on the stability of the hexagonal II (H II) phase of the phosphatidylethanolamine (PE) membrane at 20°C by small-angle X-ray scattering. As PE membrane we used DPOPE (dipalmitoleoylphosphatidylethanolamine) membrane, which was in the L α phase in 10 mM PIPES buffer (pH 7.4) at 20°C. An L α to H II phase transition occurred in the DPOPE membrane at 1.4 mM La 3+ in 0 M KCl, and at 0.4 mM La 3+ in 0.5 M KCl and above the critical concentrations the membranes were in the H II phase, indicating that La 3+ stabilizes the H II phase rather than the L α phase. The basis vector length, d, of DPOPE and DOPE (dioleoylphosphatidylethanolamine) membranes containing 16 wt% tetradecane in excess water condition did not change with an increase in La 3+ concentration, suggesting that La 3+ did not change the spontaneous curvature of these PE monolayer membranes. The chain-melting transition temperature of the dielaidoylphosphatidylethanolamine membrane increased with an increase in La 3+ concentration, indicating that the lateral compression pressure increased. To elucidate the effects of a small percentage of ‘guest’ lipids with longer acyl chains than the average length of ‘host’ lipids on the stability of the H II phase, we investigated the effects of the concentration of a guest lipid (DOPE) in a host lipid (DPOPE) membrane on their phase behavior and structure. 12 mol% DOPE induced an L α to H II phase transition in DOPE/DPOPE membrane, without changing the spontaneous curvature of the monolayer membrane. We found that Ca 2+ also induced an L α to H II phase transition in the DPOPE membrane, and compared the effects of Ca 2+ on PE membranes with those of La 3+. Based on these results, we have proposed a new model for the mechanism of the L α to H II phase transition and the stabilization of the H II phase by La 3+.

Structure and Dynamics of Selectively Deuterated Self-Assembled Silver n-Octadecanethiolate Layered Materials

The dynamics of the alkyl chains below and above the chain-melting transition temperature of long-chain silver n-alkanethiolate layered compounds were studied using variable-temperature 2H NMR and IR spectroscopies. Two specifically deuterated compounds, silver-d35-octadecanethiolate [AgSCH2(CD2)16CD3] and silver-1,1-d2-octadecanethiolate [AgSCD2(CH2)16CH3], were used to identify the behavior of different regions of the chains. The 2H NMR studies provide details about the alkyl chain motion, whereas IR spectroscopy provides details of the alkyl chain conformation. A key structural component of these materials is a polymeric −Ag−S− backbone from which the alkyl chains extend outward from both sides. The result is a two-dimensional, ribbonlike structure. The alkyl chains are highly ordered and adopt an extended all-trans conformation, which is maintained up to a few degrees below the phase transition temperature. The occurrence of a significant gauche population is observed above the phase transition temper...

Effects of alkyl chain length on the solid state phase behaviors of alkyldimethylamine oxide hydrochlorides and hemihydrochlorides

Solid phase behaviors of alkyldimethylamine oxide hydrochlorides (C nDAO·HCl) and hemihydrochlorides (C nDAO·1/2HCl) were investigated by differential scanning calorimetry (DSC) and infrared spectroscopy on five members n=10, 12, 14, 16 and 18. Two main transitions were commonly detected by DSC on the protonated (cationic) species (C nDAO·HCl): the order–disorder transition of the hydrocarbon chain (or chain melting) and the melting to a liquid crystalline(LC) phase. Both the chain-melting transition temperature T chain and the associated thermodynamic quantities Δ H chain and Δ S chain increased systematically with the chain length. Only one phase transition assigned to the partial chain melting was detected by DSC on the hemihydrochlorides (C nDAO·1/2HCl) except for n=12. The associated enthalpy changes Δ H tr were smaller than Δ H chain of the corresponding hydrochlorides by about 10–15 kJ mol −1. For the hydrochlorides, the high temperature solid phase (S2) was stable only for a limited temperature range. On the contrary, the S2 phase of hemihydrochlorides was stable over a considerable temperature range. This stable nature of the hemihydrochlorides suggested the attractive head group interaction inferred from the previous solution studies.

Mixed membranes of sphingolipids and glycerolipids as studied by spin-label ESR spectroscopy. A search for domain formation.

The temperature dependences of the ESR spectra from different positional isomers of sphingomyelin and of phosphatidylcholine spin-labeled in their acyl chain have been compared in mixed membranes composed of sphingolipids and glycerolipids. The purpose of the study was to identify the possible formation of sphingolipid-rich in-plane membrane domains. The principal mixtures that were studied contained sphingomyelin and the corresponding glycerolipid phosphatidylcholine, both from egg yolk. Other sphingolipids that were investigated were brain cerebrosides and brain gangliosides, in addition to sphingomyelins from brain and milk. The outer hyperfine splittings in the ESR spectra of sphingomyelin and of phosphatidylcholine spin-labeled on C-5 of the acyl chain were consistent with mixing of the sphingolipid and glycerolipid components, in fluid-phase membranes. In the gel phase of egg sphingomyelin and its mixtures with phosphatidylcholine, the outer hyperfine splittings of sphingomyelin spin-labeled at C-14 of the acyl chain of sphingomyelin are smaller than those of the corresponding sn-2 chain spin-labeled phosphatidylcholine. This is in contrast to the situation with sphingomyelin and phosphatidylcholine spin-labeled at C-5, for which the outer hyperfine splitting is always greater for the spin-labeled sphingomyelin. The behavior of the C-14 spin-labels is attributed to a different geometry of the acyl chain attachments of the sphingolipids and glycerolipids that is consistent with their respective crystal structures. The two-component ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled at C-14 of the acyl chain directly demonstrate a broad two-phase region with coexisting gel and fluid domains in sphingolipid mixtures with phosphatidylcholine. Domain formation in membranes composed of sphingolipids and glycerolipids alone is related primarily to the higher chain-melting transition temperature of the sphingolipid component.

Thermotropic Phase Behavior of Cationic Lipid−DNA Complexes Compared to Binary Lipid Mixtures

The thermotropic phase behavior of zwitterionic/cationic binary lipid mixtures is investigated and compared to its corresponding lipidic phase diagram of mixtures complexed with DNA. We focus on isoelectric cationic lipid-DNA condensates where the number of cationic lipids equals the number of phosphate groups on the DNA. Using differential scanning calorimetry, X-ray scattering, freeze fracture electron microscopy, and film balance, we studied mixtures of di-myristoyl-phosphatidyl-choline (DMPC) and the cationic lipid, dimyristoyl-tri-methyl-ammonium-propane (DMTAP). The lipid phase diagram shows the well-known LR, L‚′, and P‚′ ripple phase with peritectic behavior at a low molar fraction of cationic lipid, TAP < 0.12. Beyond TAP ) 0.8 crystalline phases appear. A systematic variation in the hydrocarbon chain tilt in the prevailing L‚′ phase is measured by wide-angle X-ray scattering. Most importantly, the L‚′ phase shows strong nonideal mixing with an azeotropic point at about 1:1 molar stoichiometry. This finding is related to the reduced headgroup area for equimolar mixtures found in monolayer pressure-area isotherms. The intercalation of DNA in cationic lipid-DNA complexes affects the lipid-phase behavior 2-fold: (i) the chain-melting transition temperature shifts to higher temperatures and (ii) a demixing gap with coexistence of lipid vesicles and lipid DNA complexes arises at a low cationic fraction, TAP < 0.25. In agreement with experiments we present a thermodynamic model that describes the shift of the melting transition temperatures by DNA-induced electrostatic screening of the cationic membrane.

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers.

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.

Thermodynamic Analysis of Chain-Melting Transition Temperatures for Monounsaturated Phospholipid Membranes: Dependence on cis-Monoenoic Double Bond Position

Unsaturated phospholipid is the membrane component that is essential to the dynamic environment needed for biomembrane function. The dependence of the chain-melting transition temperature, T t, of phospholipid bilayer membranes on the position, n u, of the cis double bond in the glycerophospholipid sn-2 chain can be described by an expression of the form T t = T t c(1 + h′ c| n u − n c|)/(1 + s′ c| n u − n c|), where n c is the chain position of the double bond corresponding to the minimum transition temperature, T t c, for constant diacyl lipid chain lengths. This implies that the incremental transition enthalpy (and entropy) contributed by the sn-2 chain is greater for whichever of the chain segments, above or below the double-bond position, is the longer. The critical position, n c, of the double bond is offset from the center of the sn-2 chain by an approximately constant amount, δn c ≈ 1.5 C-atom units. The dependence of the parameters T t c, h′ c, and s′ c on sn-1 and sn-2 chain lengths can be interpreted consistently when allowance is made for the chain packing mismatch between the sn-1 and sn-2 chains. The length of the sn-2 chain is reduced by ∼0.8 C-atom units by the cis double bond, in addition to a shortening by ∼1.3 C-atom units by the bent configuration at the C-2 position. Based on this analysis, a general thermodynamic expression is proposed for the dependence of the chain-melting transition temperature on the position of the cis double bond and on the sn-1 and sn-2 chain lengths. The above treatment is restricted mostly to double-bond positions close to the center of the sn-2 chain. For double bonds positioned closer to the carboxyl or terminal methyl ends of the sn-2 chain, the effects on transition enthalpy can be considerably larger. They may be interpreted by the same formalism, but with different characteristic parameters, h′ c and s′ c, such that the shorter of the chain segments makes a considerably smaller contribution to the calorimetric properties of the chain-melting transition.

Vesicle formation in aqueous dimyristoyl phosphatidylcholine suspensions: a cryo-electron microscopy study

The swelling of phospholipid in water above the chain-melting transition temperature produces lamellar structures in various shapes and sizes. These structures are potentially useful as drug, gene or cosmetic delivery systems. Cryo-electron microscopy has been used to demonstrate that the morphology of the structures in aqueous dimyristoyl phosphatidylcholine suspensions is affected by the degree of agitation. Long, undulating bilayer tubes are created, as hydrating phospholipid crystals flow through water. The proportion of unilamellar bilayer tubes to multilamellar tubes can be manipulated by varying the degree of agitation. Evidence is presented to show that agitation can transform multilamellar bilayer structures into unilamellar structures. A mechanism for the formation of unilamellar and multilamellar vesicles from these structures is discussed.

Chain-melting transition temperatures of phospholipids with acylated or alkylated headgroups ( N-acyl phosphatidylethanolamines and O-alkyl phosphatidic acids), or with α-branched chains

The biphasic dependence of the chain-melting transition temperature on chainlength, n N , of the headgroup-attached chain of N-acyl phosphatidylethanolamines and of O-alkyl phosphatidic acids is interpreted in terms of a different linear dependence of the transition enthalpy and entropy on n N for long and short chains, respectively. A consistent expression of the form T t ( n N ) = T N [| n N − Δn N | + Δh′( n o′ N − n N )]/[| n N − Δn N | + Δs′( n o′ N − n N )], where Δn N is the critical chainlength at which the packing mode of the headgroup-attached chains changes, is found to apply systematically to the transition temperatures for both sets of lipids, over the range n N = 2–18. This thermodynamic analysis demonstrates that the headgroup-attached chain is located in an environment that differs for long and short chains. Similar considerations apply also to phosphatidylcholines with a-branched glycerol-attached chains.

Temperature dependent membrane phase reorganization in giant vesicles

Giant unilamellar vesicles (GUV) from diacetylenic phospholipid, 1,2 bis (heptacosa-8,10-diynoyl)-sn-glycero-3-phosphocholine (DC 6,15PC), have been prepared by applying electric field to the aqueous dispersion maintained above its chain melting transition temperature ( T m) of 58.9°C. Previous studies have shown that large vesicles (∼1 μm or greater diameter) of the diacetylenic lipid transform into the tubule morphology when the temperature of the dispersion medium is dropped below its T m but these giant vesicles (200 μm diameter) are stable far below T m. Then within a 2° temperature range at about 32°C (26° below T m) the giant vesicles suddenly collapse, leaving oddly branched structures. Giant vesicles formed from the saturated lipid 1,2 distearoyl phosphatidylcholine, by contrast, remain intact as the temperature is lowered, eventually adhering to one another to form a gel.

Phosphatidylcholine–fatty acid membranes: effects of headgroup hydration on the phase behaviour and structural parameters of the gel and inverse hexagonal (H II) phases

The phase behaviour and structural parameters of a homologous series of saturated diacyl phosphatidylcholine/fatty acid 1:2 (mol/mol) mixtures having chain lengths from C 12 to C 20 were studied by X-ray diffraction and calorimetry, as a function of water content. The chain–melting transition temperatures of the 1:2 PC/FA mixtures are found to be largely independent of the degree of hydration. For all chain lengths, the tilted L β′ and rippled P β′ gel phases of the pure PC component are replaced by an untilted L β gel phase in the 1:2 PC/FA mixtures. This gel phase swells considerably upon hydration, with a limiting water layer thickness in the range 18–24 Å, depending on the chain length. However, unlike pure phospholipid systems, the lateral chain packing within the gel phase bilayers is essentially identical in both the dry and the fully hydrated states. The fluid bilayer L α phase is suppressed in the 1:2 mixtures, being replaced by inverse non-lamellar phases for all chain lengths greater than C 12, and at all levels of hydration. For chain lengths of C 16 and greater, the inverse hexagonal H II phase is formed directly upon chain melting, at all water contents. For the shorter chain length mixtures, the behaviour is more complex, with the H II phase forming at low hydration, but with bicontinuous cubic phases appearing at higher levels of hydration. The implications of these surprising results are explored, in terms of the effective hydrophilicity of the associated PC and FA headgroups and the packing within the interfacial region. We suggest that the presence of the fatty acids significantly alters the lateral stress profile across the lipid monolayer in the fluid state, compared to that of the corresponding pure PC system, such that inverse phases, where the interface bends towards the water, become strongly favoured. Furthermore, for short chain lengths, packing constraints favour the formation of phases with negative interfacial Gaussian curvature, such as the bicontinuous cubic phases, rather than the H II phase, which has more severe chain packing frustration.